Vaccines and immunotherapeutics using IL-28 and compositions and methods of using the same

ABSTRACT

Compositions, recombinant vaccines and live attenuated pathogens comprising one or more isolated nucleic acid molecules that encode an immunogen in combination with an isolated nucleic acid molecule that encodes IL-28 or a functional fragment thereof are disclosed. Methods of inducing an immune response in an individual against an immunogen, using such compositions are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage filing and claims priorityto PCT Application Serial No. PCT/US2009/039648, filed Apr. 6, 2009,which claims priority to U.S. Provisional Patent Application No.61/042,674, filed Apr. 4, 2008, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to improved vaccines, improved methods forprophylactically and/or therapeutically immunizing individuals againstimmunogens, and to improved immunotherapeutic compositions and improvedimmunotherapy methods.

BACKGROUND OF THE INVENTION

This application claims priority to U.S. Provisional Application No.61/042,674 filed Apr. 4, 2008, which is incorporated herein byreference.

Immunotherapy refers to modulating a person's immune responses to imparta desirable therapeutic effect. Immunotherapeutics refer to thosecompositions which, when administered to an individual, modulate theindividual's immune system sufficient to ultimately decrease symptomswhich are associated with undesirable immune responses or to ultimatelyalleviate symptoms by increasing desirable immune responses. In somecases, immunotherapy is part of a vaccination protocol in which theindividual is administered a vaccine that exposes the individual to animmunogen against which the individual generates an immune response insuch cases, the immunotherapeutic increases the immune response and/orselectively enhances a portion of the immune response (such as thecellular arm or the humoral arm) which is desirable to treat or preventthe particular condition, infection or disease.

U.S. Pat. No. 7,135,170, which is incorporated herein by reference,discloses nucleic acid and amino acid sequences of human IL-28A andhuman IL-28B and the administration of IL-28A or IL-28B protein toindividuals infected with a virus.

U.S. Pat. No. 7,491,391, which is incorporated herein by reference,discloses antibodies that bind to IL-23p19. Compositions comprising theantibodies and uses comprising administration of the antibodies incombination with other agents are described including the anti-IL-23p19antibody in combination with any interleukin protein includes IL-28.

U.S. Patent Application Publication No. 20050037018, which isincorporated herein by reference, discloses HCV vaccines in combinationwith antiviral agents to treat individuals who are infected with HCV.IL-28 protein is included in the list of antiviral compounds used totreat HCV-infected individuals in combination with the HCV vaccinesdisclosed.

U.S. Patent Application Publication No. 20060165668, which isincorporated herein by reference, discloses cancer cells transfectedwith nucleic acid molecules encoding two or more therapeutic proteins,and treating individuals who have cancer by administering such cancercells to them. Cytokines are included among the therapeutic proteinslisted and IL-28 is included in the list of cytokines.

U.S. Patent Application Publication No. 20060263368, which isincorporated herein by reference, discloses anti-cancer compounds whichincludes a cancer cell targeting moiety and a anti-cell proliferationmoiety and the use of such compounds to treat cancer and prevent orreverse chemoresistance of cancer cells. Hormones are included among thecancer cell targeting moiety listed and IL-28 is included in the list ofhormones.

U.S. Patent Application Publication No. 20070066552, which isincorporated herein by reference, discloses formulations for deliveringnucleic acid molecules that encode therapeutic proteins. IL-28 isincluded among the list of proteins described as therapeutic proteins.

Vaccine protocols can be improved by the delivery of agents thatmodulate a person's immune responses to induce an improved immuneresponse. In some vaccination protocols in which the individual isadministered a vaccine that exposes the individual to an immunogenagainst which the individual generates an immune response, an agent isprovided that increases the immune response and/or selectively enhancesa portion of the immune response (such as the cellular arm or thehumoral arm) which is desirable to treat or prevent the particularcondition, infection or disease.

Vaccines are useful to immunize individuals against target antigens suchas allergens, pathogen antigens or antigens associated with cellsinvolved in human diseases. Antigens associated with cells involved inhuman diseases include cancer-associated tumor antigens and antigensassociated with cells involved in autoimmune diseases.

In designing such vaccines, it has been recognized that vaccines thatproduce the target antigen in cells of the vaccinated individual areeffective in inducing the cellular arm of the immune system.Specifically, live attenuated vaccines, recombinant vaccines which useavirulent vectors and DNA vaccines each lead to the production ofantigens in the cell of the vaccinated individual which results ininduction of the cellular arm of the immune system. On the other hand,killed or inactivated vaccines, and sub-unit vaccines which compriseonly proteins do not induce good cellular immune responses although theydo induce an effective humoral response.

A cellular immune response is often necessary to provide protectionagainst pathogen infection and to provide effective immune-mediatedtherapy for treatment of pathogen infection, cancer or autoimmunediseases. Accordingly, vaccines that produce the target antigen in cellsof the vaccinated individual such as live attenuated vaccines,recombinant vaccines that use avirulent vectors and DNA vaccines areoften preferred.

While such vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines. There is a need forcompositions and methods that produce an enhanced immune response.

Likewise, while some immunotherapeutics are useful to modulate immuneresponse in a patient there remains a need for improvedimmunotherapeutic compositions and methods.

SUMMARY OF THE INVENTION

The present invention relates to a composition an isolated nucleic acidmolecule that encodes an immunogen in combination with an isolatednucleic acid molecule that encodes or IL-28 or functional fragmentsthereof.

The present invention further relates to a composition an isolatednucleic acid molecule that encodes both an immunogen and IL-28 orfunctional fragments thereof.

The present invention relates to injectable pharmaceutical compositionscomprising an isolated nucleic acid molecule that encodes an immunogenin combination with an isolated nucleic acid molecule that encodes IL-28or functional fragments thereof.

The present invention relates to injectable pharmaceutical compositionscomprising an isolated nucleic acid molecule that encodes both animmunogen and IL-28 or functional fragments thereof.

The present invention farther relates to methods of inducing an immuneresponse in an individual against an immunogen, comprising administeringto the individual a composition an isolated nucleic acid molecule thatencodes an immunogen in combination with an isolated nucleic acidmolecule that encodes IL-28 or functional fragments thereof.

The present invention further relates to methods of inducing an immuneresponse in an individual against an immunogen, comprising administeringto the individual a nucleic acid molecule that encodes an immunogen andIL-28 or functional fragments thereof.

The present invention further relates to recombinant vaccines comprisinga nucleotide sequence that encodes an immunogen operably linked toregulatory elements, a nucleotide sequences that encode IL-28 orfunctional fragments thereof, and to methods of inducing an immuneresponse in an individual against an immunogen comprising administeringsuch a recombinant vaccine to an individual.

The present invention further relates to a live attenuated pathogen,comprising a nucleotide sequence that encodes IL-28 or functionalfragments thereof, and to methods of inducing an immune response in anindividual against a pathogen comprising administering the liveattenuated pathogen to an individual.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows data from experiments measuring interferon gamma responsefrom mouse splenocytes when compared with mice that were immunized withthe Gag plasmid in the presence or absence of IL-28 co-treatment.

FIG. 2 shows data from analysis of splenocytes in the presence orabsence of IL-28 treatment using flow cytometry.

FIG. 3 shows data from the experiments described in Example 3. RD cellswere transfected with 3 μg of rhesus macaque IL-28B (macIL-28B) or anempty vector as a control. Supernatants were assayed for the presence ofmacaque IL-28B via ELISA 48 hours post transfection.

FIG. 4 also shows data from the experiments described in Example 3.Rhesus macaques were immunized twice with a plasmid encoding HIV Polalone or in combination with a plasmid encoding macaque IL-28B. Additionof macIL-28B increased antigen specific immune responses by ˜3 fold whenassayed by IFNgamma ELISpot.

FIGS. 5A and 5B show immunization schedules and plasmid maps. FIG. 5Aindicates that mice were immunized on Day 0 and Day 14 with amulti-clade HIV Gag construct and with or without adjuvant, followed byelectroporation using the CELLECTRA® adaptive constant current deviceafter each immunization. On Day 21 mice were sacrificed and lymphocyteswere isolated and analyzed. FIG. 5B shows plasmid maps for murine IL-28Band IL-12 constructs.

FIGS. 6A and 6B show expression and secretion of murine IL-12 and murineIL-28B in vitro. FIG. 6A shows data from Western blotting for murineIL-12p40 and murine IL-28 proteins from HEK 293T cell lysates 48 hourspost transfection. Mock transfected cells received empty pVAX vector.FIG. 6B shows data from ELISAs which show secretion of the active IL-12p35/p40 heterodimer as well as the IL-28 protein into the supernatantsof transfected cells.

FIGS. 7A and 7B show HIV Gag Specific IFNγ and IL-4 ELISpots fromIsolated Splenocytes. FIG. 7A shows the effects of cytokine adjuvants onthe induction of a Th1 response measured via the use antigen-specificIFNγ ELISpots performed on isolated splenocytes. ELISpots were performedon splenocytes harvested from mice that received IL-12 as an adjuvant orIL-28B as an adjuvant (n=4) and IFNγ spot forming units (SFU) werecounted. FIG. 7B shows the effects of cytokine adjuvants on theinduction of a Th2 response measured in the same fashion using IL-4 ELISpots.

FIGS. 8A, 8B and 8C show HIV Gag Specific IgG in sera from vaccinatedanimals. Sera from control (pVAX) or immunized animals (n=4) was assayedfor the presence of HIV Gag specific antibodies via ELISA one week postimmunization. (FIG. 8A shows total IgG. FIG. 8B shows IgG1. FIG. 8Cshows IgG2a.

FIGS. 9A and 9B show differential induction of regulatory T Cells andTGFβ secretion during immunization. The presence of Regulatory T cells(CD4+/CD25^(hi)/FoxP3+) was assayed from isolated splenocytes from allgroups (n=4) via flow cytometry (FIG. 9A). Analysis of flow cytometryshows differences in TReg populations, while analysis of cytokinesecretion from these cells shows differences in TGFβ release (FIG. 9B).p values reflect comparisons between mice vaccinated with Gag4Y alonewith mice vaccinated with Gag4Y plus IL-12 or IL-28B.

FIGS. 10A, 10B and 10C show changes in CD8+ T Cell populations,granularity and degranulation. The percentage of CD8+ T cells(CD3+/CD8+) was assessed via flow cytometry in the spleen and mesentericlymphnodes (FIG. 10A). Antigen specific induction of perforin in CD8+ Tcells was analyzed by flow cytometry via comparison of unstimulatedcells (NP) with cells stimulated with HIV Gag peptides (Peptide).Results from a single experiment are shown (FIG. 10B) and averages forall experiments are graphed (FIG. 10C). Antigen specific cytolyticdegranulation was measured via stimulation with peptide in the presenceof an antibody to CD107a, followed by analysis using flow cytometry(FIG. 10C). p values reflect comparisons between mice vaccinated withGag4Y alone with mice vaccinated with Gag4Y plus IL-12 or IL-28B.

FIGS. 11A, 11B and 11C show protection from death in a lethal Influenzachallenge. FIG. 11A shows the effects of IL-12 and IL-28 on theinduction of a Th1 response measured via the use antigen-specific IFNγELISpots performed on isolated splenocytes. FIG. 11B shows data fromexperiments in which mice (n=8) were immunized on Day 0 and Day 14 withthe influenza NP construct and with or without adjuvant, followed byelectroporation using the CELLECTRA® adaptive constant current deviceafter each immunization. On Day 42, mice were challenge intranasallywith 10 LD50 of A/PR/8/34, an H1N1 influenza strain. Mortalityassociated with influenza infection was tracked over the course of 14days (FIG. 11B).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the term “IL-28” refers to interleukin 28 protein, whichis an interferon lambda, including different variants thereof, such as,but not limited to, IL-28A, IL-28B and IL-28C.

As used herein, “functional fragment” is meant to refer to a fragment ofIL-28 that, when delivered in conjunction with an immunogen, provides anincreased immune response compared to the immune that is induced whenthe immunogen is delivered without the fragment. Fragments are generally10 or more amino acids in length.

As used herein the term “target protein” is meant to refer to peptidesand protein encoded by gene constructs of the present invention that actas target proteins for an immune response. The terms “target protein”and “immunogen” are used interchangeably and refer to a protein againstwhich an immune response can be elicited. The target protein is animmunogenic protein that shares at least an epitope with a protein fromthe pathogen or undesirable cell-type such as a cancer cell or a cellinvolved in autoimmune disease against which an immune response isdesired. The immune response directed against the target protein willprotect the individual against and/or treat the individual for thespecific infection or disease with which the target protein isassociated. In some embodiments, the target protein is a pathogenantigen such as a viral protein or fragment thereof. In someembodiments, the target protein is a viral protein or fragment thereoffrom HCV. In some embodiments, the target protein is a viral protein orfragment thereof from a virus other than HCV.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes a targetprotein or immunomodulating protein. The coding sequence includesinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of the individual to whom the nucleicacid molecule is administered.

As used herein, the term “expressible form” refers to gene constructsthat contain the necessary regulatory elements operably linked to acoding sequence that encodes a target protein or an immunomodulatingprotein, such that when present in the cell of the individual, thecoding sequence will be expressed.

As used herein, the term “sharing an epitope” refers to proteins thatcomprise at least one epitope that is identical to or substantiallysimilar to an epitope of another protein.

As used herein, the term “substantially similar epitope” is meant torefer to an epitope that has a structure that is not identical to anepitope of a protein but nonetheless invokes a cellular or humoralimmune response that cross-reacts to that protein.

As used herein, the term “intracellular pathogen” is meant to refer to avirus or pathogenic organism that, at least part of its reproductive orlife cycle, exists within a host cell and therein produces or causes tobe produced, pathogen proteins.

As used herein, the term “hyperproliferative diseases” is meant to referto those diseases and disorders characterized by hyperproliferation ofcells.

As used herein, the term “hyperproliferative-associated protein” ismeant to refer to proteins that are associated with a hyperproliferativedisease.

The invention arises from the discovery that when delivered as part of avaccine, nucleic acid molecules that encode IL-28 and functionalfragments thereof, and combinations thereof modulate immune responses.Accordingly nucleic acid molecules that encode IL-28 and functionalfragments thereof, and combinations thereof may be delivered asimmunotherapeutics in combination with or as components of a vaccine.

IL-28 proteins and nucleic acid molecules that encode such proteins aredisclosed in U.S. Pat. Nos. 7,135,170 and 7,157,559, which are eachincorporated herein by reference. In addition, U.S. Pat. Nos. 6,927,040and 7,038,032 are each incorporated herein by reference. Interferon-likeprotein Zcyto21, Kotenko et al., Nat. Immunol. 4(1):69-77, 2003 andSheppard et al., Nat. Immunol. 4(1):63-68, 2003, are also incorporatedherein by reference

GENBANK Accession numbers for the protein sequence for human IL-28A areNP_742150 and AAR24510, which are each incorporated herein by reference.

GENBANK Accession numbers for the protein sequence for human IL-28B areNP_742151 and AAR24509, which are each incorporated herein by reference.

GENBANK Accession number for the protein sequence for human IL-28C isAAQ01561, which is incorporated herein by reference.

GENBANK Accession number Q8IZJ0, which is incorporated herein byreference, refers to Interleukin-28A precursor (IL-28A) (Interferonlambda-2) (IFN-lambda-2) (Cytokine ZCYTO20).

GENBANK Accession number Q8IZ19, which is incorporated herein byreference, refers to Interleukin-28B precursor (IL-28B) (IL-28C)(Interferon lambda-3) (IFN-lambda-3) (Interferon lambda-4)(IFN-lambda-4) (Cytokine ZCYTO22).

GENBANK Accession number NM_173065, which is incorporated herein byreference, refers to Homo sapiens interleukin 28 receptor, alpha(interferon, lambda receptor) (IL28RA), transcript variant 3, mRNA.

GENBANK Accession number NM_172138, which is incorporated herein byreference, refers to Homo sapiens interleukin 28A (interferon, lambda 2)(IL28A), mRNA.

GENBANK Accession number NM_172139, which is incorporated herein byreference, refers to Homo sapiens interleukin 28B (interferon, lambda 3)(IL28B), mRNA.

GENBANK Accession number AY129153, which is incorporated herein byreference, refers to Homo sapiens interleukin 28 receptor A splicevariant 3 (IL28RA) mRNA, complete cds; alternatively spliced.

GENBANK Accession number AY129152, which is incorporated herein byreference, refers to Homo sapiens interleukin 28 receptor A splicevariant 2 (IL28RA) mRNA, complete cds; alternatively spliced.

GENBANK Accession number AY129151, which is incorporated herein byreference, refers to Homo sapiens interleukin 28 receptor A (IL28RA)mRNA, complete cds; alternatively spliced

GENBANK Accession number AY129149, which is incorporated herein byreference, refers to Homo sapiens interleukin 28B (IL28B) mRNA, completecds.

GENBANK Accession number AY129148, which is incorporated herein byreference, refers to Homo sapiens interleukin 28A (IL28A) mRNA, completecds.

According to some embodiments of the invention, the delivery of anucleic acid sequence that encodes IL-28 or functional fragmentsthereof, and combination with a nucleic acid sequence that encodes animmunogen to an individual enhances the immune response against theimmunogen. When the nucleic acid molecules that encode the transcriptionfactors are taken up by cells of the individual the nucleotide sequencesthat encode the IL-28 or functional fragments thereof, and the immunogenare expressed in the cells, the proteins are thereby delivered to theindividual. Aspects of the invention provide methods of delivering thecoding sequences of the proteins on a single nucleic acid molecule,methods of delivering the coding sequences of the proteins on differentnucleic acid molecules and methods of delivering the coding sequences ofthe proteins as part of recombinant vaccines and as part of attenuatedvaccines.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual against a pathogen or abnormal, disease-relatedcells. The vaccine may be any type of vaccine such as, a live attenuatedvaccine, a recombinant vaccine or a nucleic acid or DNA vaccine. Bydelivering nucleic acid molecules that encode an immunogen and IL-28 orfunctional fragments thereof the immune response induced by the vaccinemay be modulated. IL-28 is particularly useful when delivered via anexpressible nucleic acid molecule, such as for example as part of aplasmid or the genome of a recombinant vector or attenuated pathogen orcell. IL-28 is particularly useful when delivered prophylactically inorder to induce a protective immune response in an uninfected or diseasefree individual. IL-28B is particularly useful form of IL-28. IL-28 isparticularly useful when delivered to induce a protective immuneresponse in humans. In some embodiments, nucleic acid molecules encodingIL-28 are delivered in a cell free composition. In some embodiments,nucleic acid molecules encoding IL-28 are delivered in a compositionfree of cancer cells. In some embodiments, IL-28 or nucleic acidmolecules encoding IL-28 are administered free of any other cytokine. Insome embodiments, IL-28 or nucleic acid molecules encoding IL-28 areprovided without non-IL-28 sequences incorporated therein or linkedthereto.

Isolated cDNA that encodes the immunomodulating proteins are useful as astarting material in the construction of constructs that can producethat immunomodulating protein. Using standard techniques and readilyavailable starting materials, a nucleic acid molecule that encodes animmunomodulating protein may be prepared.

The present invention relates to compositions for delivering theimmunomodulating proteins and methods of using the same. Aspects of thepresent invention relate to nucleic acid molecules that comprise anucleotide sequence that encodes IL-28 or functional fragments thereofoperably linked to regulatory elements in combination with a nucleotidesequence that encodes an immunogen operably linked to regulatoryelements Aspects of the present invention relate to compositions whichcomprise a nucleic acid molecule that comprises a nucleotide sequencethat encodes IL-28 or functional fragments thereof operably linked toregulatory elements in combination with a nucleic acid molecule thatcomprises a nucleotide sequence that encodes an immunogen operablylinked to regulatory elements. The present invention further relates toinjectable pharmaceutical compositions that comprise such nucleic acidmolecules.

The nucleic acid molecules may be delivered using any of several wellknown technologies including DNA injection (also referred to as DNAvaccination), recombinant vectors such as recombinant adenovirus,recombinant adenovirus associated virus and recombinant vaccinia virus.

DNA vaccines are described in U.S. Pat. Nos. 5,593,972, 5,739,118,5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055,5,676,594, and the priority applications cited therein, which are eachincorporated herein by reference. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are bothincorporated herein by reference.

Routes of administration include, but are not limited to, intramuscular,intransally, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Preferred routes of administration include to mucosal tissue,intramuscular, intraperitoneal, intradermal and subcutaneous injection.Genetic constructs may be administered by means including, but notlimited to, traditional syringes, needleless injection devices, or“microprojectile bombardment gene guns”.

Another route of administration involves the use of electroporation todeliver the genetic construct, as described in U.S. Pat. Nos. 5,273,525,5,439,440, 5,702,359, 5,810,762, 5,993,434, 6,014,584, 6,055,453,6,068,650, 6,110,161, 6,120,493, 6,135,990, 6,181,964, 6,216,034,6,233,482, 6,241,701, 6,347,247, 6,418,341, 6,451,002, 6,516,223,6,567,694, 6,569,149, 6,610,044, 6,654,636, 6,678,556, 6,697,669,6,763,264, 6,778,853, 6,865,416, 6,939,862 and 6,958,060, which arehereby incorporated by reference.

Examples of electroporation devices and electroporation methodspreferred for facilitating delivery of the DNA vaccines include thosedescribed in U.S. Pat. No. 7,245,963 by Draghia-Akli, et al., U.S.Patent Pub. 2005/0052630 submitted by Smith, et al., the contents ofwhich are hereby incorporated by reference in their entirety. Alsopreferred, are electroporation devices and electroporation methods forfacilitating delivery of the DNA vaccines provided in co-pending andco-owned U.S. patent application Ser. No. 11/874,072, filed Oct. 17,2007, which claims the benefit under 35 USC 119(e) to U.S. ProvisionalApplication Ser. Nos. 60/852,149, filed Oct. 17, 2006, and 60/978,982,filed Oct. 10, 2007, all of which are hereby incorporated in theirentirety.

The following is an example of an embodiment using electroporationtechnology, and is discussed in more detail in the patent referencesdiscussed above: electroporation devices can be configured to deliver toa desired tissue of a mammal a pulse of energy producing a constantcurrent similar to a preset current input by a user. The electroporationdevice comprises an electroporation component and an electrode assemblyor handle assembly. The electroporation component can include andincorporate one or more of the various elements of the electroporationdevices, including: controller, current waveform generator, impedancetester, waveform logger, input element, status reporting element,communication port, memory component, power source, and power switch.The electroporation component can function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Insome embodiments, the electroporation component can function as morethan one element of the electroporation devices, which can be incommunication with still other elements of the electroporation devicesseparate from the electroporation component. The use of electroporationtechnology to deliver the CD28 constructs is not limited by the elementsof the electroporation devices existing as parts of oneelectromechanical or mechanical device, as the elements can function asone device or as separate elements in communication with one another.The electroporation component is capable of delivering the pulse ofenergy that produces the constant current in the desired tissue, andincludes a feedback mechanism. The electrode assembly includes anelectrode array having a plurality of electrodes in a spatialarrangement, wherein the electrode assembly receives the pulse of energyfrom the electroporation component and delivers same to the desiredtissue through the electrodes. At least one of the plurality ofelectrodes is neutral during delivery of the pulse of energy andmeasures impedance in the desired tissue and communicates the impedanceto the electroporation component. The feedback mechanism can receive themeasured impedance and can adjust the pulse of energy delivered by theelectroporation component to maintain the constant current.

In some embodiments, the plurality of electrodes can deliver the pulseof energy in a decentralized pattern. In some embodiments, the pluralityof electrodes can deliver the pulse of energy in the decentralizedpattern through the control of the electrodes under a programmedsequence, and the programmed sequence is input by a user to theelectroporation component. In some embodiments, the programmed sequencecomprises a plurality of pulses delivered in sequence, wherein eachpulse of the plurality of pulses is delivered by at least two activeelectrodes with one neutral electrode that measures impedance, andwherein a subsequent pulse of the plurality of pulses is delivered by adifferent one of at least two active electrodes with one neutralelectrode that measures impedance.

In some embodiments, the feedback mechanism is performed by eitherhardware or software. Preferably, the feedback mechanism is performed byan analog closed-loop circuit. Preferably, this feedback occurs every 50μs, 20 μs, 10 μs or 1 μs, but is preferably a real-time feedback orinstantaneous (i.e., substantially instantaneous as determined byavailable techniques for determining response time). In someembodiments, the neutral electrode measures the impedance in the desiredtissue and communicates the impedance to the feedback mechanism, and thefeedback mechanism responds to the impedance and adjusts the pulse ofenergy to maintain the constant current at a value similar to the presetcurrent. In some embodiments, the feedback mechanism maintains theconstant current continuously and instantaneously during the delivery ofthe pulse of energy.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule. DNA may beintroduced into cells, where it is present on a transient basis, in theform of a plasmid or plasmids. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may constitute part of the genetic materialin attenuated live microorganisms or recombinant microbial vectors whichare administered to subjects. Gene constructs may be part of genomes ofrecombinant viral vaccines where the genetic material remainsextrachromosomal. Genetic constructs include regulatory elementsnecessary for gene expression of a nucleic acid molecule. The elementsinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression of the sequence that encodes the target protein or theimmunomodulating protein. It is necessary that these elements beoperably linked to the sequence that encodes the desired proteins andthat the regulatory elements are operable in the individual to whom theyare administered.

An initiation codon and a stop codon are generally considered to be partof a nucleotide sequence that encodes the desired protein. However, itis necessary that these elements are functional in the individual towhom the gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV)such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals, bovinegrowth hormone polyadenylation (bgh-PolyA) signal and LTRpolyadenylation signals. in particular, the SV40 polyadenylation signalthat is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode a target protein, the immunomodulating proteinand, additionally, genes for proteins which further enhance the immuneresponse against such target proteins. Examples of such genes are thosewhich encode other cytokines and lymphokines such as alpha-interferon,gamma-interferon, platelet derived growth factor (PDGF), TNF, GM-CSF,epidermal growth factor (EGF), IL-1, IL-2, 11-4, IL-6, IL-10, IL-12 andIL-15 including IL-15 having the signal sequence deleted and optionallyincluding the signal peptide from IgE.

The compositions used in the methods may further comprise one or more ofthe following proteins and/or nucleic acid molecules encoding suchproteins, as set forth in U.S. Ser. No. 10/139,423, which corresponds toU.S. Publication No. 20030176378, which is incorporated herein byreference: Major Histocompatibility Complex antigens including MajorHistocompatibility Complex Class I antigen or Major HistocompatibilityComplex Class II antigen; death domain receptors including, but notlimited to, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR,LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; death signals,i.e. proteins that interact with the death domain receptors including,but not limited to FADD, FAP-1, TRADD, RIP, FLICE, and RAIDD; or deathsignals that include ligands that bind death domain receptors andinitiate apoptosis including, but not limited to, FAS-L, and TNF; andmediators that interact with death domain receptors including, but notlimited to, FADD, MORT1, and MyD88; toxins including proteins which killcells such as, but not limited to, insect and snake venoms, bacterialendotoxins such as Psuedomoneus endotoxin, double chain ribosomeinactivating proteins such as ricin including single chain toxin, andgelonin.

The compositions used in the methods may further comprise one or more ofthe following proteins and/or nucleic acid molecules encoding suchproteins, as set forth in U.S. Ser. No. 10/560,650, which corresponds toU.S. Publication No. 20070041941, which is incorporated herein byreference: IL-15 including fusion proteins comprising non-IL-15 signalpeptide linked to IL-15 protein sequences such as fusion proteinscomprising an IgE signal peptide linked to IL-15 protein sequences,CD40L, TRAIL; TRAILrecDRC5, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40 LIGAND, NKG2D, F461811 or MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, CD30, CD153 (CD30L), Fos, c-jun, Sp-1, Ap1, Ap-2,p38, p65Rel, MyD88, IRAK, TRAF6, IkB, NIK, SAP K, SAP1, JNK2, JNK1B2,JNK1B1, JNK2B2, JNK2B1, JNK1A2, JNK2A1, JNK3A1, JNK3A2, NF-kappa-B2, p49splice form, NF-kappa-B2, p100 splice form, NF-kappa-B2, p105 spliceform, NF-kappa-B 50 K chain precursor, NFkB p50, human IL-1.alpha.,human IL-2, human IL-4, murine IL-4, human IL-5, human IL-10, humanIL-15, human IL-18, human TNF-.alpha., human TNF-.beta., humaninterleukin 12, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF,G-CSF, GM-CSF, M-CSF, LFA-3, ICAM-3, ICAM-2, ICAM-1, PECAM, P150.95,Mac-1, LFA-1, CD34, RANTES, IL-8, MIP-1.alpha., E-selecton, CD2, MCP-1,L-selecton, P-selecton, FLT, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP,Apo-3, AIR, LARD, NGRF, DR4 (TRAIL), DR5, KILLER, TRAIL-R2, TRICK2, DR6,ICE, VLA-1, and CD86 (B7.2).

The compositions used in the methods may further comprise one or more ofthe following proteins and/or nucleic acid molecules encoding suchproteins, as set forth in U.S. Ser. No. 10/560,653, which corresponds toU.S. Publication No. 20070104686, which is incorporated herein byreference: Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK,TRAF6, IkB, Inactive NIK, SAP K, SAP-1, INK, interferon response genes,NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C,NKG2E, NKG2F, TAP1, and TAP2.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the geneticconstruct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs that are functional in the cells.

In some embodiments, gene constructs may be provided to in order toproduce coding sequences for the immunomodulatory proteins describedherein linked to IgE signal peptide.

One method of the present invention comprises the steps of administeringnucleic acid molecules intramuscularly, intranasally, intraperatoneally,subcutaneously, intradermally, or topically or by lavage to mucosaltissue selected from the group consisting of inhalation, vaginal,rectal, urethral, buccal and sublingual.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a polynucleotide function enhanceror a genetic vaccine facilitator agent. Polynucleotide functionenhancers are described in U.S. Pat. Nos. 5,593,972 and 5,962,428, whichare each incorporated herein by reference. Genetic vaccine facilitatoragents are described in U.S. Pat. No. 5,739,118, which is incorporatedherein by reference. The co-agents that are administered in conjunctionwith nucleic acid molecules may be administered as a mixture with thenucleic acid molecule or administered separately simultaneously, beforeor after administration of nucleic-acid molecules. In addition, otheragents which may function transfecting agents and/or replicating agentsand/or inflammatory agents and which may be coadministered with apolynucleotide function enhancer include growth factors, cytokines andlymphokines such as a-interferon, gamma-interferon, GM-CSF, plateletderived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1,IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15 as well as fibroblast growthfactor, surface active agents such as immune-stimulating complexes(ISCOMS), LPS analog including monophosphoryl Lipid A (WL), muramylpeptides, quinone analogs and vesicles such as squalene and squalene,and hyaluronic acid may also be used administered in conjunction withthe genetic construct In some embodiments, an immunomodulating proteinmay be used as a polynucleotide function enhancer. In some embodiments,the nucleic acid molecule is provided in association withpoly(lactide-co-glycolide) (PLG), to enhance delivery/uptake.

The pharmaceutical compositions according to the present inventioncomprise about 1 nanogram to about 2000 micrograms of DNA. In somepreferred embodiments, pharmaceutical compositions according to thepresent invention comprise about 5 nanograms to about 1000 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 100 to about200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

According to some embodiments of the invention, methods of inducingimmune responses against an immunogen are provided by delivering acombination of the immunogen and IL-28 or functional fragments thereofto an individual. The vaccine may be a live attenuated vaccine, arecombinant vaccine or a nucleic acid or DNA vaccine.

The present invention is useful to elicit enhanced immune responsesagainst a target protein, i.e. proteins specifically associated withpathogens, allergens or the individual's own “abnormal” cells. Thepresent invention is useful to immunize individuals against pathogenicagents and organisms such that an immune response against a pathogenprotein provides protective immunity against the pathogen. The presentinvention is useful to combat hyperproliferative diseases and disorderssuch as cancer by eliciting an immune response against a target proteinthat is specifically associated with the hyperproliferative cells. Thepresent invention is useful to combat autoimmune diseases and disordersby eliciting an immune response against a target protein that isspecifically associated with cells involved in the autoimmune condition.

According to some aspects of the present invention, DNA or RNA thatencodes a target protein and immunomodulating protein is introduced intothe cells of tissue of an individual where it is expressed, thusproducing the encoded proteins. The DNA or RNA sequences encoding thetarget protein and immunomodulating protein are linked to regulatoryelements necessary for expression in the cells of the individual.Regulatory elements for DNA expression include a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct.

In some embodiments, expressible forms of sequences that encode thetarget protein and expressible forms of sequences that encode bothimmunomodulating proteins are found on the same nucleic acid moleculethat is delivered to the individual.

In some embodiments, expressible forms of sequences that encode thetarget protein occur on a separate nucleic acid molecule fromexpressible forms of sequences that encode the immunomodulatory protein.In some embodiments, expressible forms of sequences that encode thetarget protein and expressible forms of sequences that encode one ormore of the immunomodulatory proteins occur on a one nucleic acidmolecule that is separate from the nucleic acid molecule that containexpressible forms of sequences that encode the immunomodulating protein.Multiple different nucleic acid molecules can be produced and deliveredaccording to the present invention.

The nucleic acid molecule(s) may be provided as plasmid DNA, the nucleicacid molecules of recombinant vectors or as part of the genetic materialprovided in an attenuated vaccine. Alternatively, in some embodiments,the target protein and immunomodulating protein may be delivered as aprotein in addition to the nucleic acid molecules that encode them orinstead of the nucleic acid molecules which encode them.

Genetic constructs may comprise a nucleotide sequence that encodes atarget protein or an immunomodulating protein operably linked toregulatory elements needed for gene expression. According to theinvention, combinations of gene constructs that include one constructthat comprises an expressible form of the nucleotide sequence thatencodes a target protein and one construct that includes an expressibleform of the nucleotide sequence that encodes an immunomodulating proteinare provided. Delivery into a living cell of the DNA or RNA molecule(s)that include the combination of gene constructs results in theexpression of the DNA or RNA and production of the target protein andone or more immunomodulating proteins. An enhanced immune responseagainst the target protein results.

The present invention may be used to immunize an individual againstpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated such as viruses, and prokaryote such as gonorrhea, listeriaand shigella. In addition, the present invention is also useful toimmunize an individual against protozoan pathogens that include a stagein the life cycle where they are intracellular pathogens. Table 1provides a listing of some of the viral families and genera for whichvaccines according to the present invention can be made. DNA constructsthat comprise DNA sequences that encode the peptides that comprise atleast an epitope identical or substantially similar to an epitopedisplayed on a pathogen antigen such as those antigens listed on thetables are useful in vaccines. Moreover, the present invention is alsouseful to immunize an individual against other pathogens includingprokaryotic and eukaryotic protozoan pathogens as well as multicellularparasites such as those listed on Table 2.

Tables

TABLE 1 Viruses Picornavirus Family Genera: Rhinoviruses: (Medical)responsible for −50% cases of the common cold. Etheroviruses: (Medical)includes polioviruses, coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus. Apthoviruses: (Veterinary)these are the foot and mouth disease viruses. Target antigens: VP1, VP2,VP3, VP4, VPG Calcivirus Family Genera: Norwalk Group of Viruses:(Medical) these viruses are an important causative agent of epidemicgastroenteritis. Togavirus Family Genera: Alphaviruses: (Medical andVeterinary) examples include Sindbis virus, RossRiver virus andVenezuelan Eastern & Western Equine encephalitis viruses. Reovirus:(Medical) Rubella virus. Flariviridae Family Examples include: (Medical)dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis andtick borne encephalitis viruses. West Nile virus (Genbank NC001563,AF533540, AF404757, AF404756, AF404755, AF404754, AF404753, AF481864,M12294, AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 andAF202541) Representative Target antigens: E NS5 C Hepatitis C Virus:(Medical) these viruses are not placed in a family yet but are believedto be either a togavirus or a flavivirus. Most similarity is withtogavirus family. Coronavirus Family: (Medical and Veterinary)Infectious bronchitis virus (poultry) Porcine transmissiblegastroenteric virus (pig) Porcine hemagglutinating encephalomyelitisvirus (pig) Feline infectious peritonitis virus (cats) Feline entericcoronavirus (cat) Canine coronavirus (dog) SARS associated coronavirusThe human respiratory coronaviruses cause about 40% of cases of commoncold. EX. 224E, OC43 Note - coronaviruses may cause non-A, B or Chepatitis Target antigens: E1 - also called M or matrix protein E2 -also called S or Spike protein E3 - also called BE orhemagglutin-elterose glycoprotein (not present in all coronaviruses) N -nucleocapsid Rhabdovirus Family Genera: Vesiculovirus, Lyssavirus:(medical and veterinary) rabies Target antigen: G protein, N proteinFiloviridae Family: (Medical) Hemorrhagic fever viruses such as Marburgand Ebola virus Paramyxovirus Family: Genera: Paramyxovirus: (Medicaland Veterinary) Mumps virus, New Castle disease virus (importantpathogen in chickens) Morbillivirus: (Medical and Veterinary) Measles,canine distemper Pneumovirus: (Medical and Veterinary) Respiratorysyncytial virus Orthomyxovirus Family (Medical) The Influenza virusBunyavirus Family Genera: Bunyavirus: (Medical) California encephalitis,La Crosse Phlebovirus: (Medical) Rift Valley Fever Hantavirus: Puremalais a hemahagin fever virus Nairvirus (Veterinary) Nairobi sheep diseaseAlso many unassigned bungaviruses Arenavirus Family (Medical) LCM, Lassafever virus Reovirus Family Genera: Reovirus: a possible human pathogenRotavirus: acute gastroenteritis in children Orbiviruses: (Medical andVeterinary) Colorado Tick fever, Lebombo (humans) equine encephalosis,blue tongue Retroyirus Family Sub-Family: Oncorivirinal: (Veterinary)(Medical) feline leukemia virus, HTLVI and HTLVII Lentivirinal: (Medicaland Veterinary) HIV, feline immunodeficiency virus, equine infections,anemia virus Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical) manyviral types associated with cancers or malignant progression ofpapilloma. Adenovirus (Medical) EX AD7, ARD., O.B. - cause respiratorydisease - some adenoviruses such as 275 cause enteritis ParvovirusFamily (Veterinary) Feline parvovirus: causes feline enteritis Felinepanleucopeniavirus Canine parvovirus Porcine parvovirus HerpesvirusFamily Sub-Family: alphaherpesviridue Genera: Simplexvirus (Medical)HSVI (Genbank X14112, NC001806), HSVII (NC001798) Varicella zoster:(Medical Veterinary) Pseudorabies varicella zoster Sub-Familybetaherpesviridae Genera: Cytomegalovirus (Medical) HCMV MuromegalovirusSub-Family. Gammaherpesviridae Genera: Lymphocryptovirus (Medical) EBV -(Burkitt's lymphoma) Poxvirus Family Sub-Family: Chordopoxviridae(Medical - Veterinary) Genera: Variola (Smallpox) Vaccinia (Cowpox)Parapoxivirus - Veterinary Auipoxvirus - Veterinary CapripoxvirusLeporipoxvirus Suipoxviru's Sub-Family: Entemopoxviridue HepadnavirusFamily Hepatitis B virus Unclassified Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneumococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella, melioidosis; salmonella; shigellosis;haemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella);streptobacillus mortiliformis and spirillum; listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria, cholera, anthrax; donovanosis(granuloma inguinale); and bartonellosis. Pathogenic anaerobic bacteriainclude: tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis; -treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlamydial infections include: mycoplasma pneumoniae;lymphogranuloma venereum; psittacosis; and perinatal chlamydialinfections. Pathogenic eukaryotes Pathogenic protozoans and helminthsand infections thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis;giardiasis; trichinosis; filariasis; schistosomiasis; nematodes;trematodes or flukes; and cestode (tapeworm) infections.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material that encodes immunogenic proteins againstwhich a protective immune response can be mounted must be included in agenetic construct as the coding sequence for the target. Because DNA andRNA are both relatively small and can be produced relatively easily, thepresent invention provides the additional advantage of allowing forvaccination with multiple pathogen antigens. The genetic construct usedin the genetic vaccine can include genetic material that encodes manypathogen antigens. For example, several viral genes may be included in asingle construct thereby providing multiple targets.

Tables 1 and 2 include lists of some of the pathogenic agents andorganisms for which genetic vaccines can be prepared to protect anindividual from infection by them. In some preferred embodiments, themethods of immunizing an individual against a pathogen are directedagainst human immunodeficiency virus (HIV), herpes simplex virus (HSV),hepatitis C virus (HCV), West Nile Virus (WNV) or hepatitis B virus(HBV).

Another aspect of the present invention provides a method of conferringa protective immune response against hyperproliferating cells that arecharacteristic in hyperproliferative diseases and to a method oftreating individuals suffering from hyperproliferative diseases.Examples of hyperproliferative diseases include all forms of cancer andpsoriasis.

It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. To immunize against hyperproliferative diseases,a genetic construct that includes a nucleotide sequence that encodes aprotein that is associated with a hyperproliferative disease isadministered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides; which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein that is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used target antigens for autoimmune disease. Othertumor-associated proteins can be used as target proteins such asproteins that are found at higher levels in tumor cells including theprotein recognized by monoclonal antibody 17-IA and folate bindingproteins or PSA.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer inindividual. Using genetic screening and/or family health histories, itis possible to predict the probability a particular individual has fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of genetic constructs serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein.

In treating or preventing cancer, embodiments which are free of cellsare particularly useful.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs) thatare involved in the disease have been characterized. These TCRs includeV.beta.-3, V.beta.-14, 20 V.beta.-17 and Va-17. Thus, vaccination with aDNA construct that encodes at least one of these proteins will elicit animmune response that will target T cells involved in RA. See: Howell, M.D., et al., 1991 Proc. Nat. Acad. Sci. USA 88:10921-10925; Piliard, X.,et al, 1991 Science 253:325-329; Williams, W. V., et al., 1992 J Clin.Invest. 90:326-333; each of which is incorporated herein by reference.In MS, several specific variable regions of TCRs that are involved inthe disease have been characterized. These TCRs include VfP and Va-10.Thus, vaccination with a DNA construct that encodes at least one ofthese proteins will elicit an immune response that will target T cellsinvolved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et al, 1990 Nature 345:344-346; each ofwhich is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs that areinvolved in the disease have been characterized. These TCRs includeV.beta.-6, V.beta.-8, V.beta.-14 and Va-16, Va-3C, Va-7, Va-14, Va-15,Va-16, Va-28 and Va-12. Thus, vaccination with a DNA construct thatencodes at least one of these proteins will elicit an immune responsethat will target T cells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes DNA constructsthat encode the variable region of such anti-DNA antibodies found in thesera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al, 1990 Proc. Natl. Acad Sci. USA87:1066, which is incorporated herein by reference.

In addition to using expressible forms of immunomodulating proteincoding sequences to improve genetic vaccines, the present inventionrelates to improved attenuated live vaccines and improved vaccines thatuse recombinant vectors to deliver foreign genes that encode antigens.Examples of attenuated live vaccines and those using recombinant vectorsto deliver foreign antigens are described in U.S. Pat. Nos. 4,722,848;5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424;5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668;5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734;5,470,734; and 5,482,713, which are each incorporated herein byreference. Gene constructs are provided which include the nucleotidesequence that encodes an IL-28 or functional fragments thereof, whereinthe nucleotide sequence is operably linked to regulatory sequences thatcan function in the vaccine to effect expression. The gene constructsare incorporated in the attenuated live vaccines and recombinantvaccines to produce improved vaccines according to the invention.

The present invention provides an improved method of immunizingindividuals that comprises the step of delivering gene constructs to thecells of individuals as part of vaccine compositions which include DNAvaccines, attenuated live vaccines and recombinant vaccines. The geneconstructs comprise a nucleotide sequence that encodes an IL-28 orfunctional fragments and that is operably linked to regulatory sequencesthat can function in the vaccine to effect expression. The improvedvaccines result in an enhanced cellular immune response.

EXAMPLES Example 1

IL-28 is a member of the newest class of interferons, InterferonLambdas, and has a heterodimeric receptor composed of IL-28Ralpha andIL-10Rbeta. As the IL-10Rbeta chain is also a part of the IL-10receptor, IL-28 was employed in vaccination studies in hopes of blockingIL-10 binding to its target receptor.

The inclusion of plasmid IL-28 with a plasmid HIV Gag constructsignificantly augmented the interferon gamma response from mousesplenocytes when compared with mice that were immunized with acomposition in which the only plasmid is the Gag plasmid alone. Micewere immunized with empty vector (pVAX), a multi-clade HIV Gag construct(HIV Gag−plasmid HIV Gag 10 μg DNA in 30 μl aqueous solution of sodiumcitrate bupivacaine) or the HIV Gag construct with an IL-28 plasmid (HIVGag IL-28−plasmid HIV Gag 10 μg DNA+plasmid IL-28 5 μg DNA in 30 μlaqueous solution of sodium citrate bupivacaine)). As shown in the datain FIG. 1, IL-28 increased the ELISpot number by greater than 7 foldwhen compared to HIV Gag alone.

Also surprisingly, analysis of splenocytes using flow cytometrysuggested that IL-28 increased splenic CD8 T cells. Mice were immunizedas above and the percentage of CD8 T cells was analyzed by flowcytometry. As shown in the data in FIG. 2, the addition of IL-28increased the percentage of CD8 T cells by 4.72% over mice immunizedwith pVAX alone, which accounts for an increase of ˜20% of total CD8s.Mice that were immunized with the HIV Gag construct alone had anincrease of only 0.48% over pVAX mice, accounting for an increase of ˜2%of total CD8s. (White bars indicate increase in CD8% over pVAX control)

Thus, while previous attempts at employing interferons as adjuvants invaccination have had disappointing results, results herein indicate thatIL-28 is an effective adjuvant in vaccination, particularly DNAvaccination.

Example 2

Mice were immunized with 1) HIV Gag construct (HIV Gag−plasmid HIV Gag10 μg DNA in 30 μl aqueous solution of sodium citrate bupivacaine) or 2)the HIV Gag construct with an IL-28 plasmid (HIV Gag IL-28−plasmid HIVGag 10 μg DNA+plasmid IL-28 3 μg DNA in 30 μl aqueous solution of sodiumcitrate bupivacaine) or the HIV Gag construct with IL-28 protein (HIVGag−plasmid HIV Gag 10 μg DNA in 30 μl aqueous solution of sodiumcitrate bupivacaine plus 40 ng IL-28 protein), or 4) the HIV Gagconstruct with interferony protein (HIV Gag−plasmid HIV Gag 10 μg DNA in30 μl aqueous solution of sodium citrate bupivacaine plus 40 nginterferony protein).

Using ELISpot, the mice receiving plasmid IL-28 showed an increasedanti-Gag immune response compared to HIV Gag alone. Neither IL-28protein nor interferon γ protein increased anti-Gag immune responsecompared to HIV Gag alone.

Example 3

While IL-28B (IFNλ3) has been suggested as being used as a potentadjuvant in mouse studies of DNA vaccination, ifs effectiveness inregards to augmenting antigen-specific immune responses has not beenstudied in larger animals, such as non-human primates. We desired totest a codon and RNA optimized plasmid encoding rhesus macaque IL-28B invaccination studies against HIV antigens as an immunoadjuvant in theseanimals. Optimization of the adjuvant plasmid in this method not onlyincreases expression of the encoded gene and stabilizes resulting RNAstructures, but also reduces the potential for integration into the hostgenome as well as eliminating any microRNAs that may have been encodedwithin the gene, resulting in a high safety profile. Analysis ofexpression of macaque IL-28B (macIL-28B) was carried out via in vitrotransfection of a rhabdosarcoma (RD) cell line with macIL-28B or anempty vector. Supernatants harvested from cells transfected withmacIL-28B, but not with empty vector, showed high quantities of macaqueIL-28B present at 48 hours post transfection when assayed via ELISA,suggesting high degrees of plasmid expression (FIG. 3).

Upon seeing high degrees of expression in vitro, we decided to addmacIL-28B to an immunization regimen against HIV Pol in rhesus macaques.Addition of IL-28B lead to a ˜3 fold increase in HIV Pol specificIFNgamma release as gauged by ELISpot after 2 immunizations (FIG. 4).These data suggest that the macIL-28B plasmid is a novel method ofexpressing high levels of IL-28B and can be used as an effectiveimmunoadjuvant in a non-human primate model of DNA vaccination.

Example 4

Improving the potency of immune responses is paramount among issuesconcerning vaccines against deadly pathogens. IL-28B belongs to thenewly described Interferon Lambda (IFNλ) family of cytokines, and hasnot yet been assessed for its potential ability to influence adaptiveimmune responses or act as a vaccine adjuvant. We compared the abilityof plasmid encoded IL-28B to boost immune responses to a multi-cladeconsensus HIV Gag plasmid during DNA vaccination with that of IL-12. Weshow here that IL-28B, like IL-12, is capable of robustly enhancingadaptive immunity. Moreover, we describe for the first time how IL-28Breduces Regulatory T cell populations during DNA vaccination, whereasIL-12 increases this cellular subset. We also show that IL-28B, unlikeIL-12, is able to increase the percentage of splenic CD8+ T cells invaccinated animals, and that these cells are more granular and havehigher antigen-specific cytolytic degranulation when compared to cellstaken from animals that received IL-12 as an adjuvant. Lastly, we reportthat IL-28B can induce 100% protection from mortality after a lethalinfluenza challenge. These data suggest that IL-28B is a strongcandidate for further studies of vaccine or immunotherapy protocols.

Introduction

Having a comprehensive understanding of the immune system and itscomponents is critical not only for understanding host-pathogeninteractions during infections, but also in the context of vaccinedevelopment and design. In regards to immune-associated signalingcompounds, such as cytokines, vaccination studies may additionally giveus a means by which to study how these molecules affect antigen specificimmune responses.

DNA vaccination is a safe and effective method of inducing antigenspecific immune responses in vivo¹⁻³ that lends itself to theintroduction of immune modulators. The ability to easily add plasmidsencoding cytokines into DNA vaccination platforms allows for thesimultaneous assessment of how a cytokine may influence adaptive immuneresponses as well as determining its potential value as a vaccineadjuvant. Furthermore, recent data in nonhuman primates with optimizedDNA formulations are showing more promising immune profiles. Improvingon these encouraging results is an important goal.

The Interferon Lambda family consists of three recently discoveredcytokines: IL-29, IL-28A and IL-28B (IFNλ1, 2, and 3, respectively)⁴⁻⁷.All three cytokines have been shown to be expressed in response to viralinfections in vitro, and are secreted primarily by dendritic cells andmacrophages⁴⁻⁷. Additionally, all three cytokines are classified asInterferons due to the fact that treatment of cells with these cytokinescan induce an antiviral state which inhibits viral replication inculture, owing to STAT, IRF and ISGF activation through the IL-28receptor⁴⁻⁷. While receptor expression has been shown on a variety ofleukocytes, including T-lymphocytes⁸, the relative ability of IL-28 toshape antigen-specific adaptive immune responses has not beenextensively studied to this point.

In this study, we have analyzed the ability of IL-28B to act as anadjuvant in a DNA vaccination setting, and compared its ability toaugment immune responses with that of IL-12, which is a potent andperhaps best established DNA immunoadjuvant⁹⁻¹³. In doing so, we havecharacterized the impact of IL-28B on the antigen specific adaptiveimmune response, which has not yet been studied. The inclusion ofplasmid encoded IL-28B or IL-12 lead to increased antigen specificcellular immune responses over vaccination with antigen alone, as gaugedby IFNγ ELISpot and detection of perforin by flow cytometry. IL-28B, butnot IL-12, was further able to increase antigen-specific IgG2a, antigenspecific cytolytic degranulation and the percentage of CD8+T cells foundin the spleen. Additionally, we found that the IL-28B adjuvant reducedthe number of CD4+/CD25^(hi)/FoxP3+(Treg) cells found in the spleens ofvaccinated animals, while IL-12 increased the size of this population.Lastly, we show here, when used as an adjuvant for vaccination in mice,IL-28B is able to augment immune responses in such a fashion so as toresult in 100% protection from death after a lethal influenza challenge.This study shows that IL-28B may act as an effective adjuvant forcellular immunity in vivo and is the first to describe the differentialaffects of IL-28B and IL-12 on Treg populations after DNA vaccination.This constitutes the first major analysis of the ability of IL-28B toshape adaptive immune responses in vivo.

Materials and Methods

Plasmids.

The IL-12 plasmid encoding murine p35 and p40 proteins has beendescribed^(11,14). Murine IL-28B had a high efficiency leader sequenceadded to the 5′ end of the gene and was synthesized, codon optimized andsubsequently subcloned into the pVAX1 backbone by GeneArt (Renensberg,Germany). Plasmids expressing HIV-1 Gag (Gag4Y) were prepared aspreviously described¹⁵.

Animals.

All animals were housed in a temperature-controlled, light-cycledfacility at the University of Pennsylvania, and their care was under theguidelines of the National Institutes of Health and the University ofPennsylvania. All animal experiments were performed in accordance withnational and institutional guidelines for animal care and were approvedby the Institutional Review Board of the University of Pennsylvania.

Immunization of Mice.

The quadriceps muscle of 8 week old female BALB/c mice (JacksonLaboratory) were injected 2 times, 2 weeks apart, and electroporated aspreviously described¹⁶ using the CELLECTRA® adaptive constant currentdevice (VGX Pharmaceuticals, The Woodlands, Tex.). For experiments inmice, the animals (n=4 or 8 per group) were immunized with either 10 μgof pVAX1 or 10 μg of HIV-1 Gag (Gag4Y) or Influenza NP (NP) alone orwith varying amounts of murine IL-12 or murine IL-28B plasmid, dependingon the experiment. Co-administration of various gene plasmids involvedmixing the designated DNA plasmids before injection in 0.25%bupivicaine-HCL (Sigma) in isotonic citrate buffer to a final volume of30 μl.

ELISpot.

Both IFN-γ and IL-4 ELISpot was performed to determine antigen specificcytokine secretion from immunized mice. ELISpots were carried out permanufacturers protocols (R&D Systems) using 96-well plates (Millipore).2×10⁵ splenocytes from immunized mice were added to each well of theplate and stimulated overnight at 37° C., 5% CO₂, in the presence of R10(negative control), concanavalin A (positive control), or specificpeptide (HIV-1 Gag) antigens (10 μg/ml). HIV-1 Consensus Gag Clade C15-mer peptides spanning the entire protein, overlapping by 11 aminoacids, were acquired from the AIDS Reagent and Reference Repository(Frederick, Md.).

Cell Culture and Staining for Flow Cytometry.

Splenocytes harvested from immunized mice were washed and thenresuspended with R10 media to a final concentration of 10⁷ cells/ml.Cells were seeded into 96 well plates in a volume of 100 μl and anadditional 100 μl of media alone (negative control), media containingHIV-1 Gag Consensus Clade C peptides or media containing PMA andIonomycin (positive control) was then added and plates were place at 37°C. In cultures being used to measure degranulation, anti-CD107a PE wasadded at this time as an enhanced stain. Cultures used to measureintracellular perforin levels did not receive this antibody. For thesecultures, ten minutes after the addition of media, peptide orPMA/Ionomycin, Mg⁺² and EGTA were added to cultures to a finalconcentration of 6 mM and 8 mM, respectively to inhibitcalcium-dependent cytolytic degranulation¹⁷. All cultures were allowedto incubate at 37° C. for 6 hours. At the end of this incubation period,plates were spun down and washed twice with PBS. Cells were then stainedwith a violet dye for viability (LIVE/DEAD Violet Viability Dye,Invitrogen) for 10 minutes at 37° C. After washing as above with PBS,cells were stained externally with anti-CD4 PerCPCy5.5 (BD Bioscience)and anti-CD8 APCCy7 (BD Bioscience) at 4° C., followed by fixing andpermeabilization (Cytofix/Cytoperm Kit, BD Bioscience). Anti-CD3 PE-Cy5(BD Bioscience) and anti-Perforin APC (eBioscience) were added and cellswere incubated again at 4° C. Cells were given a final wash with PBS andfixed in PFA at a final concentration of 1%. For flow cytometryinvolving CD4+/CD25^(hi)/FoxP3+ cells, the Mouse Regulatory T Cellstaining kit was employed (eBioscience). External staining was carriedout as above with anti-CD4 FITC and anti-CD25 APC. Fixation,permeabilization and internal staining were also carried out as aboveusing anti-FoxP3 PE.

Influenza Challenge. 28 days post immunization, anaesthetized mice wereintranasally inoculated with 10 LD50 of A/Puerto Rico/8/34 in 30 μlPBS¹⁶. All murine challenge groups were comprised of 8 mice per group.After challenge, clinical signs and mortality were recorded daily for 14days.

Statistics. Data are presented as the mean±Standard Error of the Mean(SE) calculated from data collected from at least three independentexperiments. Where appropriate, the statistical difference betweenimmunization groups was assessed by using a paired Student's t Test andyielded a specific p value for each experimental group. Comparisonsbetween samples with a p value <0.05 were considered to be statisticallydifferent and therefore significant.

Results

Plasmids Encoding Murine IL-12 and IL-28B Express and Secrete Protein

There is a constant need for discovery of new and improved adjuvants forvaccination against various viral pathogens. In the past, IL-12 has beenshown to be a potent adjuvant when employed in vaccination studies⁹⁻¹³.IL-28B has not yet been used for this purpose. In order to compare therelative abilities of these cytokines to augment antigen-specific immuneresponses, we constructed plasmids encoding murine IL-12¹¹ and murineIL-28B for use in our DNA vaccination studies (FIGS. 5A and 5B). Toconfirm whether these constructs expressed IL-12 and IL-28B, we testedthem in vitro via transfection of HEK 293T cells with 3 μg of plasmid.Cells were subsequently lysed and lysates were used in Western Blots totest for protein expression. Blots for murine IL-12p40 and murine IL-28Bproteins show that both constructs are expressed well in vitro (FIG.6A). To examine cytokine secretion to the extracellular environment,cell supernatants were obtained 48 hours post transfection. ELISAs todetect the active IL-12 p35/p40 heterodimer and the IL-28B protein fromtransfected cell culture supernatants were carried out. As shown in FIG.6B, IL-12 and IL-28B were both observed to be present at a concentrationof roughly 10,000 pg/ml in transfected culture supernatants. Uponconfirming the expression and release of both cytokines from transfectedcells, we began vaccination studies to test the ability of thesecytokines to adjuvant antigen-specific immune responses.

IL-28B Adjuvants HIV Gag-Specific IFNγ Release after Vaccination

In order to determine whether or not IL-28B had the potential tofunction as an immunoadjuvant, we used it in DNA vaccination studies incombination with a plasmid encoding a multi-clade consensus HIV-1 Gagprotein (Gag4Y) as our target antigen. In order to have a measure ofcomparison of the potency of the adjuvant affects of IL-28B, we comparedit to a cytokine that is frequently employed as an adjuvant invaccinations due to the fact that it has previously been shown to havevery potent immunoadjuvant affects: IL-12⁹⁻¹³. To that end, groups of 8week old Balb/c mice (n=4 per group) were immunized intramuscularly inthe right rear quadricep with 10 μg of empty pVAX vector (control) or 10μg of HIV Gag4Y construct alone, followed by electroporation. Additionalgroups received 10 μg of HIV Gag4Y in combination with either IL-28B orIL-12 at varying doses, also followed by electroporation. Results of anIFNγ ELISpot assay show that while immunization with Gag4Y alone wasable to induce a cellular immune response in the mice (˜400 SFU permillion splenocytes), inclusion of IL-28B was able to further increaseGag-specific IFNγ release at all doses tested several fold (FIG. 7A).Optimal adjuvant affects of IL-28B (3 to 4-fold over Gag4Y alone) wereseen at a range of 7 to 9 μg (FIG. 7A), leading us to use this dose forfurther experiments. IL-12 also increased Gag-specific IFNγ release inthis assay by just over 3-fold in the same range of doses as IL-28B(FIG. 7A). Analysis of each assay showed that responses were mediatedpredominantly by CD8+ T cells (>85% total response) and that thisprofile was not influenced by the presence of absence of adjuvant duringvaccination (data not shown). These results suggest that IL-28B can,indeed, be used to bolster antigen specific immune responses duringvaccination, with increases in IFNγ release comparable or greater thanthose seen with IL-12 which is an established, potent immunoadjuvant.

Upon confirming that IL-28B could be used to increase the cellularimmune response via increased release of the Th1-associated cytokineIFNγ, we next endeavored to determine if this adjuvant could affect therelease of a prototypical Th2 cytokine. Thus we employed an IL-4 ELISpotin the same fashion as above to observe how IL-28B might be influencingthe release of this Th2 associated cytokine. Interestingly, theinclusion of IL-12 in vaccination resulted in increases in Gag-specificIL-4 release at all doses tested, ranging from ˜200 to ˜600 SFU (FIG.7B). The optimal doses for IL-12 in the IFNγ ELISpot assay resulted in˜400 to ˜450 SFU in the IL-4 ELISpot assay, while the inclusion ofIL-28B did not show this type of affect. Instead, inclusion of IL-28B invaccination resulted in IL-4 release that was quite similar to the HIVGag4Y construct alone (FIG. 7B), suggesting that IL-28B does notincrease IL-4 release contaminant with increased IFNγ release at thesedoses. Thus, IL-28B may be thought of as inducing a more “pure”Th1-associated cytokine profile during vaccination when compared withIL-12 in that it induces IFNγ (Th1 associated) release but not IL-4 (Th2associated) release.

IL-28B but not IL-12 Increases HIV Gag Specific IgG2a

As an effective vaccination against a viral pathogen may necessitateboth a cellular and humoral immune response, we decided to examine therelative abilities of IL-28B and IL-12 to augment the level ofcirculating HIV Gag-specific antibodies when employed as adjuvantsduring vaccination. In order to accomplish this, we tested sera takenfrom immunized mice in antigen-specific ELISAs. Inclusion of IL-12 orIL-28B in conjunction with the HIV Gag4Y construct resulted in markedlydifferent antibody responses, as shown in FIG. 8. In regards to totalGag-specific IgG, immunization with the Gag4Y construct together withthe IL-28B construct lead to a small increase in levels ofantigen-specific antibodies when compared to immunization with Gag4Yalone at the lowest dilution tested (1:25) (FIG. 8A). However, inclusionof IL-12 with Gag4Y immunization actively suppressed antigen-specificIgG, with values reading very similar to those of control (pVAX) mice.This affect of IL-12 is a phenomenon that has been reported previouslyin DNA vaccination¹⁴, and is supported in the current study as well. Wenext examined different subtypes of IgG, including IgG1 and IgG2a todetermine additional effects on immune polarization. The IgG1 isotype isassociated with Th2 skewing in mice while IgG2a is associated with Th1skewing¹⁸. Regardless of the inclusion of adjuvant, DNA vaccination didnot seem to augment Gag-specific IgG1 antibody levels in any group inour assay (FIG. 8B). However, the inclusion of IL-28B in vaccinationlead to a greater than 2-fold increases in IgG2a when compared with serafrom mice vaccinated with HIV Gag4Y alone (FIG. 8C). Additionally, IL-12continued to suppress antibody responses in this assay, as evidenced bythe fact that no increase in IgG2a was seen in the IL-12 group whencompared to the control (pVAX) group. Thus IL-28B seems to be able toincrease antigen specific humoral immune responses in a heavilyTh1-biased fashion, which is in agreement with its affect on thecellular immune response (FIGS. 7A and 7B).

IL-28B Decreases Splenic CD4+/CD25^(hi)/FoxP3+ Cells, While IL-12Increases Them

IL-28B is a member of the newly described IFN λ family, and thus is alsoknown as IFN λ3¹⁸⁻²¹. Other members of the IFN λ family include IL-28A(IFN λ2) and IL-29 (IFN λ1)¹⁸⁻²¹. A previous study has suggested thatIL-29 may play a role in immune suppression and tolerance in that it maydrive dendritic cells to specifically induce the proliferation ofCD25^(hi)/FoxP3+ CD4+ T cells (Treg cells) in response to IL-2⁵. Theinduction or expansion of Treg cells could be considered a drawback tovaccination strategies within certain settings, and the ability ofIL-28B to influence this subpopulation of CD4+ T cells has notpreviously been studied. As IL-28B falls into the same IFN family asIL-29, we addressed the possibility that it may exert similar affects onthe Treg cell population. Additionally, we looked at the ability ofIL-12 to affect Tregs in this type of vaccination setting, which has notbeen previously examined.

By looking at the expression of CD4, CD25 and FoxP3 via flow cytometry(FIG. 9A), we were able to study the impact of vaccination with andwithout cytokine adjuvants on Treg populations in immunized mice. Theresults of this analysis show that immunization with the HIV Gag4Yconstruct alone resulted in a small but not statistically significantdecrease in the percentage of splenic Tregs from vaccinated mice (FIG.10B). This result is consistent with a previous report describing asimilar change in Treg populations after vaccination²⁰. Inclusion ofcytokine adjuvants in vaccination dramatically altered Treg populationsin varying fashions. Interestingly, the employment of IL-12 as animmunoadjuvant significantly increased the number of splenic Tregs inimmunized mice when compared with mice vaccinated with the HIV Gag4Yconstruct alone (FIG. 9B). This is the first time this phenomenon hasbeen reported in a vaccination setting and may constitute a previouslyunrealized phenotype of IL-12 as an adjuvant for immunization. Also ofconsiderable interest was the fact that the inclusion of IL-28B as animmunoadjuvant for vaccination caused a statistically significantdecrease in the number of splenic Tregs when compared to vaccinationwith HIV Gag4Y alone (FIG. 9B). This is the first time this ability ofIL-28B has been described and may be viewed as significant benefit ofthis cytokine when it is used as an adjuvant for vaccination. Moreover,it suggests the possibility that while they are in the same IFN family,there may be key differences between IL-28B and IL-29.

Splenocytes from Mice that Received IL-28B Secrete Less TGFβ

We reasoned that it was possible that the inclusion of IL-12 or IL-28Bin vaccination may have been phenotypically altering normal CD4+ T cellsto look like Tregs, instead of inducing the expansion of fullyfunctional Tregulatory cells. Therefore, in order to determine if thesecells were functioning as Tregs in addition to phenotypically resemblingTregs, we measured the ability of splenocytes from vaccinated mice toproduce TGFβ, which is recognized as one of the major mediators ofTreg-based non-contact immunosuppression²¹. To accomplish this, wecultured splenocytes from each group of mice for 48 hours with acombination of PMA and lonomycin in order to determine TGFβ release fromactivated cells. At the end of this time period, supernatants were takenfrom cell cultures and were used in ELISAs to detect TGFβ. As shown inFIG. 9B, activation of splenocytes isolated from mice that receivedIL-12 as an adjuvant resulted in a statistically significant increase inTGFP production when compared to mice that received Gag4Y alone (FIG.9B). This result suggests that the differences in Treg numbers observedvia flow cytometry between mice that received IL-12 and mice thatreceived HIV Gag4Y alone are correctly identifying Treg populations.Additionally, splenocytes isolated from mice that received IL-28B as anadjuvant produced significantly less TGF β when activated by PMA andlonomycin (FIG. 9B). This, again, supports the flow cytometry datasuggesting that there are differences in Treg populations in mice thatreceived IL-28B compared to mice that received HIV Gag4Y alone.

As IL-2 is a key cytokine in the induction and expansion of Tregs²¹, wereasoned that differences in Treg populations between vaccinated groupscould be due to differential production of IL-2. In order to test thispossibility we again measured cytokine release from activatedsplenocytes isolated from each group. Analysis of IL-2 output from thesecells show no significant difference between any of the groups (data notshown), suggesting that there is an alternate mechanism responsible fordifferences in Treg populations seen after DNA vaccination.

IL-28B but not IL-12 Increases Splenic CD8+ T Cells

Upon determining that IL-28B could affect the amount of splenic Tregs,we decided to investigate whether or not this adjuvant had similaraffects on other cell types after vaccination. To that end, splenocytesfrom control and vaccinated mice were analyzed for the presence of CD8 Tcells (CD3+/CD8+) by flow cytometry. As shown in FIG. 10A, thepercentage of CD8 T cells in the spleens of control (pVAX) mice were notsignificantly different than mice who received the Gag4Y constructalone, or the Gag4Y construct with the IL-12 adjuvant. However, micethat received IL-28B as an adjuvant showed significantly higherpercentages of CD8 T cells in the spleen when compared with all othergroups, suggesting that IL-28B has the ability to expand the splenicCD8+ T cell population after immunization. In order to determine whetherthis affect of IL-28B was restricted to the spleen or could be seen inother lymphoid organs and peripheral blood, we next analyzed lymphocytesisolated from the mesenteric lymph nodes (MLN) as well as circulatingPBMCs from each group of mice. Mice immunized with the HIV Gag4Yconstruct alone showed a small but not statistically significantincrease in the number of CD8+ T cells found in MLN when compared tocontrol mice. Mice that received IL-12 as an adjuvant showed increasednumbers of CD8+ T cells in MLN when compared to mice that received Gag4Yalone, and this increase was able to reach statistical significance(FIG. 10A). Mice that received IL-28B in conjunction with the HIV Gag4Yconstruct during immunization had slightly higher increases in CD8+ Tcell percentages, which reached an impressive statistical significance(p<0.005). Lymphocytes isolated from peripheral blood showed increasesin CD8+ T cell populations only in the group that received the IL-28Badjuvant, which is reminiscent of the pattern seen in the spleen (FIG.10A). These results indicate that IL-28B, but not IL-12, increases thesize of CD8+ T cell populations in the spleen and peripheral blood ofimmunized mice while both adjuvants are able to increase CD8+ T cells inMLN.

IL-28B Significantly Increases HIV Gag Specific CD8+ T Cell PerforinInduction and Degranulation

Upon determining that IL-28B was having a significant impact on thepercentage of splenic CD8+ T cells after vaccination, we decided toperform further analysis on this cellular subset. It has previously beenshown that IL-12 may influence the granularity of CD8+ T cells¹⁹. As ourprevious experiments suggested that IL-28B was having a strong influenceon cellular immunity (via IFNγ release) that was equal to or greaterthan IL-12 (FIG. 7A), we asked whether or not IL-28B could influencecell granularity in the same fashion as IL-12. Therefore, we designedexperiments to measure the antigen-specific induction of perforin inCD8+ T cells isolated from the spleens of each group of mice. In orderto determine the amount of antigen-specific perforin upregulation, weincubated isolated splenocytes with a media control or with a set ofoverlapping HIV Gag Clade C peptides for 6 hours, followed by extra- andintracellular staining for cellular markers and perforin, followed byanalysis via flow cytometry (FIG. 10B). In order to prevent cytolyticdegranulation, EGTA and Mg⁺² were added to cultures as described inMethods¹⁷. The results of this stimulation are presented in FIG. 10C.CD8+ T cells in splenocytes from all groups incubated with media aloneshowed roughly equivalent amounts of perforin, suggesting thatvaccination, with or without adjuvants, had no significant affect onbasal CD8+ T cell granularity in this system. Stimulation of splenocyteswith overlapping HIV Gag peptides showed different results. CD8+ T cellsin splenocytes from mice immunized with the HIV Gag4Y construct aloneshowed a modest increase in the percentage of cells falling into thePerforin^(hi) gate (FIG. 10B) when compared to control mice. However,CD8+ T cells taken from mice that had received IL-12 or IL-28B bothshowed increases in Perforin cell percentages that were clearly higherthan those taken from mice that received the Gag 4Y construct alone(FIG. 10C). This result is consistent with previous reports that IL-12may increase the perforin content of lymphocytes²² and is the first timethis affect of has been reported for IL-28B.

With the knowledge that IL-28B could influence the perforin content ofCD8+ T cells, we examined if this cytokine adjuvant could be affectingantigen-specific degranulation as well. To test this, we incubated cellsin the same fashion as we did to measure perforin induction, save forthe fact that no EGTA or Mg⁺² were added to cultures, and an antibody toCD107a, which is a marker for degranulation²³, was instead added at thetime of peptide stimulation as an enhanced stain. Cells were againsubjected to staining for cellular markers, followed by analysis viaflow cytometry. We observed that CD8+ T cells from mice that hadreceived the Gag 4Y construct alone showed a low level of Gag-specificdegranulation (FIG. 10C). CD8+ T cells from mice that received IL-12 incombination with the HIV Gag 4Y construct showed a modest increase inantigen-specific degranulation when compared with mice that received theGag construct alone, but this difference did not reach statisticalsignificance. However, CD8+ T cells from mice that received the IL-28Bplasmid as an adjuvant in vaccination showed a significant increase inHIV Gag-specific degranulation when compared with T cells taken frommice that did not receive adjuvant (FIG. 10C). The results show thatIL-28B causes a major and statistically significant increase in CD8+ Tcell degranulation when used as an adjuvant in DNA vaccination.

IL-28B Protects from a Lethal Influenza Challenge In Vivo

Since our assays for cellular immune responses suggested that IL-28B hadthe potential to act as a strong adjuvant for Th1 biased cellularimmunity, we decided to test the ability of this cytokine to protectagainst a lethal viral challenge in vivo. In order to accomplish this,we immunized 4 additional sets of mice (n=8 mice per group) in the samefashion as above, followed by electroporation. Control mice received 10μg of empty pVAX vector, while the other groups of mice received 10 μgof a plasmid encoding the influenza nucleoprotein (NP) alone or inaddition to IL-12 or IL-28B. The nucleoprotein of influenza is aninternal structural protein, and not exposed on the outside of thevirion. Thus, immunity to influenza infection that is targeted againstthe NP protein is cellular, as apposed to humoral immunityl⁶. Analysisof cellular immunity of vaccinated mice via IFNγ ELISpot showed that theIL-12 and IL-28B adjuvants induced increased responses to the InfluenzaNP antigen in much the same way it augmented responses to the HIV Gag4Yconstruct (FIG. 11A). After a 4 week rest period following immunization(FIG. 11B), all groups of mice were challenged intranasally with 10 LD50of an H1N1 influenza strain: A/Puerto Rico/8/34 (A/PR/8/34). The micewere monitored over the course of the next 14 days for mortalityassociated with viral infection. Results of this experiment show thatchallenge of control mice resulted in 100% mortality by Day 8 postinfection (FIG. 11C). Mice that received 10 μg of the NP constructshowed 50% mortality over the following 14 days, suggesting that the NPconstruct alone was not completely sufficient to induce protection.IL-12, when used as an adjuvant in challenge studies, has been shown inthe past to be able to induce significant protection against mortalityassociated with viral infections^(13,14). This is also the case in thecurrent study, as exhibited by the fact that mice that received IL-12 asan adjuvant to NP showed 100% protection from death as a result ofinfection. Additionally, in agreement with our previous assayssuggesting that IL-28B could induce potent cellular immune responses,mice who received IL-28B as an adjuvant to the NP construct also showed100% survival after viral challenge. The results of this experiment showthat IL-28B, when used as an adjuvant during DNA vaccination, may induce100% protection from mortality associated with viral infection in vivo.

Discussion

The study presented here shows that plasmid encoded IL-28B may havepotent affects on antigen-specific immune responses when used as anadjuvant in DNA vaccination. IL-28B was able to augment antigen-specificimmune response to a multi-clade HIV Gag antigen in a Th1-biasedfashion, which was evidenced by greatly enhanced IFNγ release duringantigen-specific ELISpots as well as increased Gag-specific IgG2a levelsdetected in the sera of vaccinated mice. Additionally, this is the firstreport to describe the ability of IL-28B to reduce splenic Tregpopulations after DNA vaccination, a great potential benefit of thiscytokine. IL-28B was also shown here to be able to expand the splenicCD8+ T cell population, and that these cells showed increased perforininduction and degranulation in response to cognate antigen. The factthat IL-28B was able to augment protection of mice in a lethal viralchallenge model makes a strong case for continued testing of thiscytokine as an adjuvant in vaccination.

The impact of IL-28B was measured against IL-12 due to the fact thatIL-12 is known to be a highly potent cytokine that is used often as anadjuvant in vaccination studies⁹⁻¹³. Analysis of this comparison showsIL-28B to be at least as potent as IL-12 in some assays, if not better.Moreover, IL-28B affords additional benefits for vaccination that IL-12does not, including increased antigen-specific antibody titers and anincreased splenic CD8+ T cell population that is capable of higherdegrees of antigen-specific cytolytic degranulation. The affects ofIL-28B and IL-12 on Treg populations was dramatically different. This isthe first study to analyze the induction of Tregs in response to IL-12in DNA vaccination and to report that this adjuvant may increase thiscell population. While the impact of this finding is not as yet clear,this result may support that IL-28B could be superior in specificsituations where cellular immunity is paramount. The specific mechanismby which mice that received IL-12 had larger Treg populations remainsunclear, although a recent report has highlighted the importance of theIL-12 receptor for the generation of Tregs in vivo²⁴, suggesting apossible link between this cytokine and the induction and expansion ofthe Treg population. The ability of IL-28B to reduce Treg numbers seemsmore likely to be a targeted mechanism, as it is able to increase somesubsets of T cells (CD8) while reducing others (Treg). It is possiblethat this, too, is mediated through the IL-28 receptor, althoughadditional studies into specific mechanisms are needed.

Results presented here include constitute a significant analysis of thefunction of IL-28B in vivo and contribute to the beginning of ourunderstanding of how IL-28B affects immune responses. The data suggeststhat IL-28B may be a regulator of the adaptive immune response inaddition to its IFN-like functions, and this affect seems to focuslargely on the number and function of CD8+ T cells. The fact that IL-28Binduces an antiviral state in addition to being able to shape antigenspecific-immune responses suggests that it is has the unique ability tobridge the gap between innate and adaptive immunity. Furthermore IL-28Bmay have a unique role in immune therapy approaches over IL-12 inspecific adjuvant settings. In particular as an adjuvant in tumorimmunity where tolerance is particularly an issue, IL-28B may be veryuseful. Further studies are needed in order to properly verify this.

REFERENCES

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The invention claimed is:
 1. A recombinant immunogenic pharmaceuticalcomposition comprising an immunogenic amount of an isolated nucleic acidconstruct comprising a nucleotide sequence that encodes a viral proteinoperably linked to regulatory elements, wherein said nucleic acidconstruct further comprises a nucleotide sequence that encodes an IgEsignal peptide and a codon-optimized and RNA-optimized nucleotidesequence that encodes IL-28B or a functional fragment thereof, whereinsaid viral protein is from a virus selected from the group consistingof: influenza virus, human immunodeficiency virus, hepatitis C virus,West Nile Virus and hepatitis B virus.
 2. An injectable immunogenicpharmaceutical composition comprising the composition of claim
 1. 3. Therecombinant immunogenic pharmaceutical composition of claim 1, whereinthe viral protein is from an influenza virus.
 4. The recombinantimmunogenic pharmaceutical composition of claim 1, wherein the viralprotein is from HIV.
 5. The recombinant immunogenic pharmaceuticalcomposition of claim 4 wherein said recombinant pharmaceuticalcomposition comprises a recombinant vaccinia vector.
 6. A method ofinducing an immune response in an individual against a viral proteinfrom a virus selected from the group consisting of: influenza virus,human immunodeficiency virus, hepatitis C virus, West Nile Virus andhepatitis B virus comprising administering to said individual arecombinant vaccine of claim
 1. 7. A live attenuated virus comprising anisolated nucleic acid construct comprising a nucleotide sequence thatencodes a viral protein operably linked to regulatory elements, whereinsaid nucleic acid construct further comprises a nucleotide sequence thatencodes an IgE signal peptide and a codon-optimized and RNA-optimizednucleotide sequence that encodes IL-28B or a functional fragmentthereof.
 8. A method of immunizing an individual against a pathogencomprising administering to said individual the live attenuated pathogenof claim
 7. 9. The recombinant immunogenic pharmaceutical composition ofclaim 1, wherein the nucleotide sequence that encodes IL-28B or afunctional fragment thereof is operably linked to a promoter.
 10. Therecombinant immunogenic pharmaceutical composition of claim 9, whereinthe promoter is a SV40 promoter, MMTV promoter, L TR promoter, or CMVimmediate early promoter.
 11. The live attenuated virus of claim 7,wherein the virus is an influenza virus.
 12. The live attenuated virusof claim 7, wherein the virus is human immunodeficiency virus, hepatitisC virus, West Nile Virus or a hepatitis B virus.
 13. An immunogenicpharmaceutical composition comprising an immunogenic amount of a nucleicacid construct comprising a nucleotide sequence that encodes a viralprotein operably linked to regulatory elements, wherein said nucleicacid construct further comprises a nucleotide sequence that encodes anIgE signal peptide and a codon-optimized and RNA-optimized nucleotidesequence that encodes IL-28B or a functional fragment thereof.
 14. Theimmunogenic pharmaceutical composition of claim 13, wherein the viralprotein is from a virus selected from the group consisting of: influenzavirus, human immunodeficiency virus, hepatitis C virus, West Nile Virusand hepatitis B virus.
 15. An injectable immunogenic pharmaceuticalcomposition comprising the pharmaceutical composition of claim
 13. 16. Arecombinant immunogenic pharmaceutical composition comprising animmunogenic amount of an isolated nucleic acid construct comprising anucleotide sequence that encodes a viral protein operably linked toregulatory elements, wherein said viral protein is from a virus selectedfrom the group consisting of: influenza virus, human immunodeficiencyvirus, hepatitis C virus, West Nile Virus and hepatitis B virus; and animmunogenic amount of an isolated nucleic acid construct encoding an IgEsignal peptide and a codon-optimized and RNA-optimized immunomodulatingprotein, wherein the immunomodulating protein consists of IL28B or afragment thereof.